Biomedical subjects

S Matzku

Publications and source records attributed to S Matzku.

At least 55 records · Page 3Linked to original sources

PubMed1989

Antigenic differences between metastatic and nonmetastatic BSp73 rat tumor variants characterized by monoclonal antibodies.

Variants AS and ASML of the BSp73 rat tumor differ markedly with respect to morphology and to the capacity for spontaneous metastasis via the lymphatics. Monoclonal antibodies (MAbs) were raised in order to identify surface molecules associated with both phenotypes. Mice were immunized with either whole cells or isolated membranes and hybridoma supernatants were screened according to the criteria of selective binding to cultured cells of the variant used for immunization. Of two MAbs reacting with the nonmetastasizing AS variant, one showed immunostaining of AS tissue. From 11 MAbs binding to the metastatic ASML variant, 6 showed specific staining of ASML tissue, while the remaining 5 MAbs showed cross-reaction with normal rat tissues. According to Western blot data, the 6 MAbs selectively binding to ASML tissue identified at least 2 different antigens, one of them showing up as a complex of 12 bands.

Animals↗

PubMed1989

Melanoma targeting with a cocktail of monoclonal antibodies to distinct determinants of the human HMW-MAA.

The monoclonal antibodies (MoAbs) 149.53, 225.28, and 763.74 which recognize distinct and spatially distant determinants of the human high molecular weight-melanoma associated antigen (HMW-MAA) do not influence the binding of each other to cultured human melanoma cells. In vitro incubation of melanoma cells with a combination of the three 125I-labeled anti-HMW-MAA MoAbs results in a marked additive binding only when the MoAbs are used at saturating concentrations. Injection of the combination of the three 125I-labeled MoAbs (up to 300 micrograms per mouse) into human melanoma-bearing nude mice does not increase the amount of radioactivity specifically localized in melanoma lesions above the level observed upon injection of corresponding doses of individual MoAbs. These results may reflect the low concentration of MoAbs which reaches tumor lesions in vivo. Therefore, administration of combinations of MoAbs to distinct determinants of HMW-MAA may not increase the sensitivity of immunoscintigraphy to visualize lesions in patients with melanoma.

Animals↗

PubMed1989

Depression of nonadaptive immunity after surgical stress: influence on metastatic spread.

The question of increased tumor cell dissemination after surgical stress was addressed in the model system of the spontaneously metastasizing rat adenocarcinoma BSp73ASML, wherein amputation of the hind leg 7 days after intrafootpad implantation of tumor cells cured the animals, while surgical stress by laparotomy 2 days prior to amputation resulted in lung metastases in 80% of rats. A detailed in vitro analysis of natural killer (NK) and macrophage (Mo) activity in different lymphatic compartments revealed the following impacts of surgery: splenic NK cells displayed unaltered activity. Yet, there was a considerable decrease in the number of lymphoid cells during the first 4 days after surgery, being followed by an overshooting repopulation. In the peripheral blood, activity levels of NK cells dropped significantly during the first 24 h after surgery; later on, NK cells appeared activated with an over 2-fold increase in lytic units (LU), 4 days after surgery, NK activity had returned towards normal levels. Most dramatic changes were observed in the peritoneal cavity, being directly involved in the surgical intervention. Six hours after surgery the peritoneal cavity was nearly depleted of NK cells and Mo, the few remaining cells being highly activated. Within 2 days the peritoneal cavity was repopulated with a 3-4 fold excess of lymphoid cells and Mo, but the repopulating cells were extremely low in lytic activity. It is concluded that depression of nonadaptive immunity after surgical stress is mainly due to traffic and repopulation with immature cells, i.e. there was no indication of suppressor cell activity. This was confirmed by a combined treatment consisting in a systemic application of Corynebacterium parvum 2 days before surgery and a local application of C. parvum after surgery, which counter-balanced the stress-induced depression of NK and Mo activity. Accelerated and increased metastatic spread could be prevented concomitantly.

Animals↗

PubMed1989

Stability in biological fluids and clearance of immunoliposomes.

The in vivo and in vitro stability of liposomes having covalently bound monoclonal antibodies (MAB) connected to their surface via a disulfide bridge (about 6 anti-melanoma MAB-molecules per liposome) in tissue fluids was investigated. These immunoliposomes were composed of equimolar amounts of hydrogenated soybean lecithin and cholesterol and had a mean diameter of 113 nm. The protein-lipid binding, change of vesicle size and the release of encapsulated carboxyfluorescein (CF) were estimated. After i.v. injection into mice, an initial protein cleavage of 21% occurred within 30 min p.a., followed by a slow elimination of intact MAB-liposomes from blood circulation (t50 = 2.2 h). These liposomes had a similar elimination rate as protein-free liposomes. The in vitro results after incubation of MAB-liposomes in blood, plasma, serum and tissue fluid were very similar with regard to the protein-lipid cleavage, but the time dependence was different. The CF-release in vitro was slower (about 1.5%/d) than the protein cleavage; the vesicle size of MAB-liposomes increased substantially in contrast to protein-free liposomes in serum during 4 days.

Animals↗

PubMed1988

Antibody targeting to the murine lymphoma ESb-MP: increased accumulation due to reduced internalization into lymphoma cells as compared to normal lymphoid cells.

Using rat monoclonal antibody (MAb) 12-15A against the spontaneously metastasizing mouse lymphoma variant ESb-MP, we elaborated conditions for targeting. In vitro, high binding of labelled antibody to ESb-MP cells and low binding to lymphoid cells (e.g., spleen cells) was noted. In vivo, we observed pronounced accumulation in spleen, lymph nodes and bone marrow, the uptake kinetics indicating high accessibility of the target antigen in these tissues, and rapid clearance of radioactivity from blood and most normal tissues, indicating degradation of the antibody and excretion of the label. Binding to lymphoma tissue was slow but persistent, resulting in high tumor:tissue ratios only after 2-3 days. Biodistribution could be dramatically changed by pre-treatment of animals with excess cold antibody, which reduced trapping of labelled antibody in normal lymphatic tissue, leading to prolonged persistence in the blood and preferential uptake into tumor tissue. Monovalent 12-15A fragments showed less pronounced binding to lymphatic tissue, while being rapidly cleared from the circulation by virtue of their inherent tendency to bind to kidney tissue. Tumor:tissue ratios up to 56:1 were obtained by a combination of pre-treatment with unlabelled 12-15A IgG or Fab fragment, followed by injection of labelled fragment or IgG, respectively. This is interpreted on the basis of differences in the internalization and retention of antigen-antibody complexes. Pre-treatment obviously leads to a temporary blockade or removal of the target antigen, which is much more efficient with normal lymphoid cells than with tumor cells. Thus, it may become possible to target antibodies into the tumor despite concurrent antigen expression on normal tissue.

Animals↗

PubMed1988

Dynamics of antibody transport and internalization.

By following the interaction of labelled anti-tumor antibodies with target cells, it was found that in most systems cross-linking of externally bound antibody by anti-IgG leads to nearly complete internalization, whereas excess unlabelled antibody induces fast release of radioactivity from the cell. Uptake in tumor tissue did not depend on presentation of antigen at the cell surface, as demonstrated with antibody RA96 directed against an intra- and inter-cellular antigen. With respect to permeation into solid tissue, autoradiography revealed highly non-homogeneous distribution of intact IgG, while antibody fragments tended to show a more homogeneous penetration. Small spontaneous metastases showed markedly higher accumulation than large tumor processes.

Animals↗

PubMed1988

Biodistribution of 131I-labelled monoclonal antibodies in human colon tumours by an ex vivo perfusion model.

We describe a model for the evaluation of anti-tumour antibody specificity, using a human carcinoma-bearing colon segment. After resection of the human colon tumour, the supplying artery was cannulated and perfused with fresh frozen plasma and heparin. Continuous control of pressure, flow, temperature, pH and various metabolic parameters were performed after administration of 131I-labelled anti-CEA antibody. Highly differentiated adenocarcinomas of the colon showed a much higher antibody uptake than undifferentiated tumours. Between 3 and 7% of the injected antibody was found in the tumour tissue. Autoradiography showed non-homogeneous binding in the tumour tissue. The non-specific antibody perfusion showed no tumour binding. We conclude that the ex vivo perfusion of resected colon carcinomas can be used to measure the kinetics of binding and clearance of MAbs in tumour tissue by direct scintigraphy. The cellular biodistribution of the antibody can be documented by means of autoradiography.

Antibodies, Monoclonal↗

PubMed1988

Optimisation of the covalent binding of monoclonal antibodies to liposomes.

Liposomes with covalently attached monoclonal antibody (mAb) may be useful for the active targeting of drugs, e.g. cytostatics, to tumor processes. For pharmacokinetic reasons the coupling reaction should be optimized with regard to the loading capacity, the number of IgG-molecules per liposome (N). The relative immunoreactivity of bound versus free mAb (r/r*) and the stability in biological fluids should be maximal, the particle size (phi) should be minimal. Furthermore, the coupling yield of the mAb should be high. This investigation was based on a previously described method, using N-hydroxysuccinimidyl-dithiopropionate for coupling anti-melanoma mAbs to liposomes composed of hydrogenated soybean lecithin, cholesterol and a dipalmitoyl-phosphatidylethanolamine derivative. Carboxyfluoresceine was used as a marker for the encapsulated phase. By systematic variation of 14 different reaction conditions a product with the following properties was obtained: N = 5, r/r* = 0.4 and phi = 120 nm. The average coupling yield was 39%. The specificity of liposomally bound mAb as assayed in in vitro tests was found to be unchanged.

Animals↗

PubMed1987

Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue.

Immunohistology provides a necessary but insufficient criterion for selecting monoclonal antibodies (MAbs) capable of tumour targeting in vivo. Additional selection procedures have been evaluated using a panel of anti-melanoma MAbs, including immunoreactivity of (labelled) MAbs, antibody affinity, kinetics of binding and release, apparent antigen density and accumulation in nude mouse transplants. According to these criteria, MAbs M.2.7.6 and M.2.9.4 showed the most favourable properties, i.e. high immunoreactivity and pronounced internalization into melanoma cells. With MAbs M.2.10.15 and KG 6-56, moderate immunoreactivity and a binding pattern characterized by temperature dependence in the absence of internalization was observed. According to the paired label assay, all four MAbs showed specific accumulation into solid melanoma tissue. However, application in the patient still requires evaluation of the side effects of antigen cross-expression on normal human tissues.

Antibodies, Monoclonal↗

PubMed1987

Modulation of melanoma-associated antigens by monoclonal antibodies as visualized by radioimmunoelectron microscopy and radioantibody binding assay.

There is a wealth of information about monoclonal antibody (MAb) specificity and function on fixed tissues, yet little is known about formation and release of antigen-antibody complexes and their functional behavior in vivo. We analyzed the pathway of radiolabeled MAbs directed against melanoma-associated antigens by radioimmunoelectron microscopy (RIEM) on metabolically active cells of the melanoma cell lines SK-MEL-28, MeWo and Colo 38 at different time intervals. In parallel, binding and release of MAbs were investigated by the radioantibody binding assay (RBA). Both procedures gave essentially concordant results. Preferentially stable binding of immune complexes (ICs) to the cell surface after 30 and 120 min was shown for the MAb L10. Internalization was demonstrated for the MAb M.2.9.4. At the ultrastructural level, direct evidence of this phenomenon was obtained by visualization of radioactivity within the cytoplasm after 120 min. In the RBA this process was indicated by resistance of bound MAbs to acid buffer desorption. RIEM pointed to different transport mechanisms: constitutive internalization by endocytotic vesicles, or receptor-mediated endocytosis by coated vesicles. Shedding was indicated for the MAb R24 by release of the ICs from the cell membrane. It was demonstrated that stable fixation of ICs on the cell surface or modulation by internalization led to high accumulation rates, while shedding of antigen-antibody complexes resulted in a low accumulation of the MAb in tumor cells. Assuming that the potential of MAbs for clinical application is determined by the biological behavior of antigen-antibody complexes, these methods are suitable for demonstration of antigenic modulation by MAbs and eventually enable us to predict the localization, penetration and distribution pattern of individual MAbs in the melanoma patient.

Antibodies, Monoclonal↗

PubMed1987

Iodination of monoclonal IgG antibodies at a sub-stoichiometric level: immunoreactivity changes related to the site of iodine incorporation.

Thirteen monoclonal antibodies (MAbs) were labeled with 125I to a different degree such as to cover the range from 0.5-20 microCi/micrograms. By SDS polyacrylamide gel electrophoresis, the amount of iodine incorporated into heavy (h) and light (l) chains was determined. Comparing different MAbs, h:1 ratios varied from 0.6-34.6, but virtually no variation was observed with individual MAbs labeled at different levels. Immunoreactivity of labeled MAbs was analyzed with antigen-positive tumor cells according to the Lineweaver Burk method. Immunoreactive fractions were found to decrease with increasing iodine incorporation in 9/12 MAbs, while binding affinities decreased in 5/12 MAbs; only 1 MAb was stable in both respects. Immunoreactivity changes were not linked to preferential h or 1 chain labeling, nor to the isotype. This result indicated incorporation of the first iodine atom to take place at individually distinct residues, with a minimum estimate of two or four sites, depending on whether preferential chain labeling or random incorporation took place. In cases where increasing labeling led to a gradual decrease of binding affinity, a shift in the spectrum of acceptor residues has to be assumed.

Animals↗

PubMed1987

What's new in cancer monitoring?

Cancer monitoring by in vitro procedures is intimately linked to the identification of suitable tumor markers. Application of monoclonal antibodies (MAbs) not only led to the definition of new markers, but also to considerable progress in analytical methodology. In addition, monoclonal antibodies may be used for tumor targeting, thus opening the way for new diagnostic procedures (radioimmunolocalization) or therapeutic approaches.

Antibodies, Monoclonal↗

PubMed1986

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part I. Chemistry and labeling technique.

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N'-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tightly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37 degrees C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

Animals↗

PubMed1986

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part II. Comparison of immunoreactivity and biodistribution of monoclonal antibodies labeled with the 67Ga-chelate or with 131I.

Coupling of the 67Ga-P-EDDHA chelate via carbodiimide to the anti-melanoma monoclonal antibody (Mab) M.2.9.4 resulted in a low degree of oligomerization, but a considerable degree of intra-molecular (inter-chain) cross-linking. However, this did not impair immunoreactivity, nor did the half-life in vivo differ substantially from that of 131I-M.2.9.4. Biodistribution analysis in normal mice showed Ga:I ratios near 1 in the blood and other tissues not involved in degradation and label excretion. In tissues of the reticulo-endothelial system (RES) and the kidneys, Ga:I ratios up to 2.51 were reached within 4 days of administration. In antigen-positive MeWo tumor tissue, retention of 67Ga also excreted that of 131I, so that tumor; organ ratios (except tumor:liver) were superior for the 67Ga-labeled MAb. It is concluded that the method of coupling pre-established 67Ga-P-EDDHA chelate to antibody results in a functionally intact tracer molecule, whose persistence in vivo is not significantly impaired. The major difference to I-labeled MAbs may be a prolonged retention of Ga in tissues (cells) physiologically involved in antibody catabolism.

Animals↗

PubMed1986

Modes of binding and internalization of monoclonal antibodies to human melanoma cell lines.

The process of monoclonal antibody (MAb) binding to tumor cells is greatly influenced by the biology of the respective antigen. This was concluded from an analysis of binding and release of MAbs and MAb fragments to melanoma cells at different concentration levels and different temperatures. With an antigen known to be stably expressed at the cell surface (i.e., Mr 97,000 protein) rapid binding of MAbs was observed at both 0 degrees C and 37 degrees C, and this was reversed by treatment with isoosmolar acid buffer. With another group of antigens, MAb binding increased continuously up to considerable levels at 37 degrees C, but not at 0 degrees C. Concomitantly, the portion of radioactive MAb not desorbable by acid buffer treatment increased, pointing to temperature-dependent internalization. With still another group of (glycolipid) antigens, the highest MAb binding was obtained with fixed cells at 0 degrees C. In this situation MAb release was particularly rapid, thus pointing to a shedding process.

Antibodies, Monoclonal↗

PubMed1985

11C-butanol for imaging of the blood-flow distribution in tumor-bearing animals.

1-11C-n-Butanol produced semiautomatically using a cyclotron was employed to investigate the whole-body distribution and kinetics of the label of this compound. Following the administration of 11C-butanol into the aorta of two dogs, more than 80% of the activity was cleared from the blood within 1 min. The activity distribution mirrored the cardiac output distribution as determined using 121I microspheres. Within 25 min p.i., a significant release of decay-corrected activity was only observed for the liver, spleen, and kidneys. Muscle and whole-body activity showed constant levels during this period. In 45 tumor transplants from rats, the dynamic behavior of the label was studied. The tissue retention of activity following injection into the a. femoralis was approximately 100% during the 1st 15 s for both tumor and muscle (n = 6). The activity release by tumors during the 1st 10 min after intra-aortic injection was 18% +/- 4.5% (n = 39; decay corrected). In five different tumor lines (n = 10), the initial 11C-butanol uptake was related to that of muscle, and the results were correlated with the tumor-to-muscle retention of 121I-microspheres (r = 0.89). In 17 tumors, the correlation between 11C-butanol uptake and the washout rate of 133Xe resulted in a correlation coefficient of 0.96. Tumor-to-muscle uptake ratios could be equally determined using intra-aortic and intravenous injection, as evaluated by intraindividual comparison in 12 rats (y = 0.01 + 0.98x; r = 0.98). 11C-Butanol appears to be an appropriate radiotracer for the assessment of blood supply to malignant tumors relative to muscle.

1-Butanol↗

PubMed1985

Immunoreactivity of monoclonal anti-melanoma antibodies in relation to the amount of radioactive iodine substituted to the antibody molecule.

The damage to monoclonal anti-melanoma antibodies caused by iodination was investigated by comparing the results obtained using the chloramine-T method and the 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenyl-glycoluril (IODOGEN) method at different levels of iodine substitution to the molecule. The level of substitution at which losses in immunoreactivity occurred was evaluated in each monoclonal antibody (MAb) studied. This phenomenon was not dependent on the method of substitution, provided that mild conditions of reaction were used. Lineweaver-Burk plots and--in cases of alterations in binding affinity--Scatchard plots were found to provide an adequate description of the binding behaviour of individual MAbs after labelling. Immunoreactivity was shown to be determined not only by the proportion of bona fide reactive MAb molecules, but also by a substitution-dependent decrease in affinity constants. The practical consequences of altered binding parameters were demonstrated by quantitating specific antibody accumulation in melanoma transplants in vivo.

Animals↗

PubMed1985

Clonal analysis of diversity in the BSp73 rat tumor.

By implantation of BSp73 ascites cells in a subcutaneous site and subsequent subcutaneous passage of either the local tumor node or metastatic lung tissue, variants were obtained which differed with respect to morphology and to metastatic capacity. The highly metastasizing variant ASML showed spherical morphology in culture, while the nonmetastatic variant AS showed adhesion and spreading. Upon cloning it was observed that colonies with fully expressed morphotypes were readily obtained from solid tissue of both variants. Parental ascites as well as the tumor line derived from the primary solid tumor gave rise to stable expression of either morphotype only after prolonged culturing. Mixing of established clones did not result in an interclonal adaptation of growth rates in vivo. Further characterization of variants AS and ASML revealed marked differences in the outer cell surface. Adhesion of AS cells onto plastic was found to be mediated by fibronectin, laminin and 4 out of 5 collagen types. ASML cells showed adhesion only with collagen type III at higher concentrations. Cytogenetic analysis revealed that the adaptation of BSp73 cells to ascitic growth ultimately led to an increase in chromosome numbers, and this was conserved in ASML cells (modal number 63, range 49-74). AS cells on the other hand showed a modal number of 47 (range 45-49). The chromosome count distribution was rather narrow in ascites cells in vivo, but it was very broad in clones derived thereof, indicating that diversity was obtained in culture rather than in vivo. The data are compatible with the assumption that the nonmetastatic variant was not preexisting in BSp73 ascites but represents a stable phenotype which infrequently arises in a particular microenvironment by chromosome loss from a hyperdiploid parental population.

Animals↗